Macroscopic membranes play an important role in many biological processes at both the cellular and organismic level. In addition, membranes are used in a number of medical, research, and industrial applications. Physiologically compatible membranes would be especially valuable for biomedical products. At present, the self-assembly of peptides into macroscopic membranes has not been reported.
A small peptide termed EAK16 (AEAEAKAKAEAEAKAK310-325 of SEQ.ID NO:2) was discovered serendipitously to self-assemble into stable macroscopic membranes in the presence of millimolar concentrations of salt. This invention relates to the self-assembly of peptides into stable macroscopic membranes. Peptides which form membranes are characterized as being amphiphilic, i.e., having alternating hydrophobic and hydrophilic amino acid residues; greater than 12 amino acids, and preferably at least 16 amino acids; complementary and structurally compatible. Complementary refers to the ability of the peptides to interact through ionized pairs and/or hydrogen bonds which form between their hydrophilic side-chains, and structurally compatible refers to the ability of complementary peptides to maintain a constant distance between their peptide backbones. Peptides having these properties participate in intermolecular interactions which result in the formation and stabilization of xcex2-sheets at the secondary structure level and interwoven filaments at the tertiary structure level.
Both homogeneous and heterogeneous mixtures of peptides characterized by the above-mentioned properties can form stable macroscopic membranes. Peptides which are self-complementary and self-compatible can form membranes in a homogeneous mixture. Heterogeneous peptides, including those which cannot form membranes in homogeneous solutions, which are complementary and/or structurally compatible with each other can also self-assemble into macroscopic membranes.
Peptides which can self-assemble into macroscopic membranes, the conditions under which membrane formation occurs, and methods for producing the membranes are described and included in this invention.
Macroscopic membranes formed of the peptide EAK16 have been found to be stable in aqueous solution, in serum, and in ethanol and are highly resistant to degradation by heat, alkaline and acidic pH (i.e., stable at pH 1.5-11), chemical denaturants (e.g., guanidine-HCl, urea and sodium dodecyl sulfate), and proteases (e.g., trypsin, xcex1-chymotrypsin, papain, protease K, and pronase). The membranes have also been found to be non-cytotoxic. The membranes are thin, transparent and resemble high density felt under high magnification. Being composed primarily of protein, the membranes can be digested and metabolized in animals and people. They have a simple composition, are permeable, and are easy and relatively inexpensive to produce in large quantities. The membranes can also be produced and stored in a sterile condition. Thus, the macroscopic membranes provided by this invention are potentially useful as biomaterial for medical products, as vehicles for slow-diffusion drug delivery, as separation matrices, and for other uses requiring permeable and water-insoluble material.
Furthermore, the salt-induced assembly of the peptides into insoluble and protease-resistant protein filaments with a xcex2-sheet secondary structure is similar in some respects to the formation of the neurofibrillary filaments and amyloid plaques associated with Alzheimer""s disease and the formation of scrapie prion protein filaments. The formation of the macroscopic membranes can therefore be useful as a model system to study these pathological processes. For example, such a model system can be used to identify drugs which inhibit filament formation and are thus potentially useful for treating Alzheimer""s disease and scrapie infection.
Peptide EAK16 was derived from a region of a yeast protein, zuotin, which exhibits a high affinity for DNA in the left-handed Z conformation. Zuotin was identified by a gel shift assay for Z-DNA binding proteins developed by the Applicants. Applicants further cloned and sequenced the gene encoding zuotin. Characterization of zuotin revealed that the protein is a potential substrate for several protein kinases and identified a putative DNA-binding domain. This invention also includes all or biologically active portions of the zuotin protein and DNA encoding zuotin.